Project Summary/Abstract Research Component 1. Alcohol addiction is a dynamic multiphasic disorder that is characterized, in part, by repeated binge/intoxication episodes where the powerful positive reinforcing effects of the drug predominate. Although multiple neural systems regulate alcohol reinforcement, we have shown that repetitive binge episodes engage glutamatergic ?-amino-3-hydroxy-5-methyl-4-isooxazole receptor (AMPAR) mechanisms of synaptic plasticity in brain reward pathways. Specifically, binge-like operant alcohol self-administration (SA) is associated with a shift toward GluA2-lacking Ca2+-permeable (CP-AMPAR) activity (increased GluA1-S831 phosphorylation and reduced GluA2 protein expression) in the basolateral amygdala (BLA). Preliminary optogenetic data show that these alcohol-induced adaptations are associated with increased AMPAR synaptic activity in nucleus accumbens core (AcbC) neurons receiving projections from BLA; thus, strongly implicating the BLA?AcbC neural circuit. We have also shown that amygdala AMPAR activity is required for the positive reinforcing effects of alcohol, and activation of amygdala AMPARs promotes escalated operant alcohol SA. This convergence of molecular, physiological, and bi-directional behavioral data support the overall hypothesis that: CP-AMPAR activity in the BLA?AcbC pathway functionally regulates the positive reinforcing effects of alcohol. We propose to test this hypothesis in three separate but integrated aims using male and female C57Bl/6J mice. First, molecular studies are proposed to evaluate AMPAR subunit protein expression (GluA1 and GluA2) and phosphorylation (pGluA1-S831 and S845) in amygdala and accumbens subregions to determine if alcohol SA produces a shift toward GluA2-lacking CP-AMPAR activity in this reward pathway. Magnetic resonance imaging (MRI) will also be coupled with an optogenetic strategy to identify specific BLA projections that are altered by alcohol SA. Second, to assess alcohol-induced adaptations in CP-AMPAR synaptic activity, we will evaluate synaptic properties of BLA neurons projecting to AcbC, and use optogenetics to test BLA?AcbC circuit function in M/F C57BL/6J mice. We predict increased CP-AMPAR signaling in BLA and BLA?AcbC circuit of alcohol SA mice. Third, site-specific pharmacological studies will determine if CP- AMPAR activity in the BLA or AcbC is necessary for alcohol reinforcement and/or sufficient for escalated binge-like SA. A site-specific AAV approach will express a dominant negative form of the AMPAR GluA1 subunit (GluA1ct) to determine if activity dependent GluA1 membrane trafficking in the BLA or AcbC regulates the reinforcing effects of alcohol. These studies will elucidate novel molecular mechanism(s) of alcohol?s reinforcing effects, which has potential to lead to new therapeutic strategies for treating behavioral pathologies associated with alcohol addiction. The overall goal of the UNC ARC is to increase understanding of molecular and cellular pathogenesis in alcoholism. To address this goal, Research Component 1 seeks to elucidate novel molecular targets of initial binge-like alcohol SA that, in turn, gain control over reinforcement processes to drive repetitive use and escalated intake, two of the most significant behavioral pathologies in alcohol addiction.